Hepatitis C virus (HCV) infection continues to be a major burden on public health, affecting approximately 170 million people worldwide and 3-4 million Americans. HCV-associated end-stage liver disease is the leading indicator of liver transplantation. Current standard therapy with pegylated interferon-? in combination with ribavirin only achieves limited (<50%) antiviral response and causes severe side effects. The HCV protease- and polymerase-specific inhibitors currently in clinical trials are promising but are undermined by rapid emergence of drug-resistant HCV mutants. Future antiviral therapy for hepatitis C likely requires a combination of several drugs targeting different steps of the HCV life cycle. The lack of knowledge about the molecular details of the HCV life cycle has significantly impeded the discovery and development of antiviral drugs against HCV infection. A more complete understanding of the roles of viral and cellular proteins in the HCV life cycle will provide additional novel targets for anti-HCV drug discovery. Our recent studies have demonstrated that human apolipoprotein E (apoE) is an important determinant for the outcomes of HCV infection and assembly. ApoE was also found to interact with the HCV NS5A in our preliminary studies. In Specific Aim 1, we will dissect apoE domains and amino acid residues important for its dual functions in HCV infection and assembly. We will also determine the importance and the underlying molecular mechanisms of apoE and its interaction with NS5A in HCV infection and assembly, respectively. In preliminary studies, we have demonstrated that adaptive mutations in the HCV non-structural (NS) proteins greatly enhanced HCV production and cytopathogenicity in cell culture. We have also found that HCV NS proteins interact with viral structural proteins, as demonstrated by experiments with the mammalian two-hybrid system and co-immunoprecipitation assay. Consistent with our findings, recent genetic studies done by others suggest that HCV NS proteins play important roles in HCV assembly and/or production. However, the underlying molecular mechanisms of the HCV NS proteins in the HCV life cycle have not been defined. In Specific Aim 2, we will decipher the roles and mechanisms of action of cell culture adaptive mutations in HCV assembly and induction of cytopathogenicity. In Specific Aim 3, we will determine the importance of viral NS proteins and their interactions with structural proteins in HCV assembly. We will accomplish these specific aims using a robust HCV reverse genetics system recently developed in our lab in conjunction with mutagenesis studies and the use of biochemical, immunological, and cell biological approaches. New knowledge derived from these studies will lead to a paradigm shift in virology with respect to the roles of viral NS proteins in HCV assembly. Our studies will also provide novel targets for discovery of antiviral drugs to effectively treat hepatitis C.